During inhalational anthrax, considerable evidence indicates that alveolar macrophages (m?s) contribute to the development of disseminated infection and disease by transporting dormant B. anthracis (Ba) spores, previously inhaled into the alveolar spaces of the lungs, to the bloodstream. Although the initial steps of spore uptake into m?s have been extensively studied, a major gap in knowledge, which is the focus of this exploratory R21 application, is the mechanism by which intracellular Ba escape infected m?s as a requisite step prior to extracellular dissemination. Here, we propose studies that challenge an existing model that predicts, analogous to several different intracellular pathogens, m? death must precede Ba release to the external environment. Instead, our preliminary data indicate that subsequent to phagocytic uptake of dormant spores into Bacillus-containing vacuoles (BCVs), a substantial fraction of intracellular Ba escape in the absence of m? killing. Because we find no evidence that Ba depart BCVs into the cytosol of infected m?s, we propose that these intracellular vacuoles represent specialized niches that are important for Ba escape. In support of this idea, infection with Ba enhances transport of lysosomal contents to the cell surface, and, induces the time-dependent enrichment of BCVs with the membrane vesicle fusion protein, synaptotagmin-7, consistent with an exocytic process. Based on these preliminary data, we will evaluate the overall hypothesis that non-lytic escape of intracellular Ba from infected m?s requires remodeling of BCVs from degradative to exocytic compartments. We will identify changes in BCV-associated proteins during the course of Ba infection, and, characterize their functional importance for the maturation of BCVs into compartments associated with egress of intracellular Ba back to the extracellular environment. These studies will provide the first detailed characterization of the intracellular niche occupied by Ba spores, and contribute to our understanding of the fundamental biology underlying inhalational anthrax.